Biology subjects

Sahid, S.

Publications and source records attributed to Sahid, S..

6 recordsLinked to original sources

bioRxiv2023

Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid ({beta}-ODAP) has restricted its cultivation. Although various cultivars with low {beta}-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal {beta}-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the {beta}-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the backcrossed mutant lines and they displayed no phenotypic abnormalities. Interestingly, the seed {beta}-ODAP content ranged between 0.001 % - 0.002 % of dry weight which is 99 % lower than the wild-type. Together, our study reports the development of transgene-free, genome-edited grass peas with insignificant levels of {beta}-ODAP in seeds for safer food in the future.

plant biology↗

bioRxiv2022

Glutathione activates PHT1;5 gene via WRKY75 transcription factor to regulate phosphate homeostasis in Arabidopsis

Phosphorus (P) is essential for plant growth, yet inorganic phosphate (Pi) is often limiting in soil. Plants respond to Pi limitation by activating P transporters and starvation genes, which boost Pi uptake or redistribute P. While glutathione (GSH) regulates iron and zinc homeostasis in Arabidopsis, its role in P homeostasis under Pi limitation remains elusive. Here, we show that GSH-S-nitrosoglutathione (GSNO) module regulates source-to-sink P allocation in Arabidopsis by transcriptionally activating the phloem-localised transporter AtPHT1;5 via the transcription factor AtWRKY75. GSH-deficient mutants, cad2-1 and pad2-1, exhibited sensitivity to Pi-starvation with altered root system architecture, reduced anthocyanin accumulation, and disrupted root-to-shoot P transport. AtPHT1;5 transcript levels were markedly reduced in these mutants but were positively responsive to exogenous GSH or GSNO. The pht1;5 mutant failed to respond to GSH treatments, demonstrating that AtPHT1;5 is essential for GSH-mediated P mobilisation. Pharmacological and genetic manipulation of nitric oxide (NO)/GSNO levels implicated GSNO as an essential intermediary. In depth molecular analyses identified AtWRKY75 as a Pi- and GSH/GSNO-responsive activator that binds w-box motifs in the AtPHT1;5 promoter. This was further supported by the lack of AtPHT1;5 induction in the wrky75 mutants. Finally, AtWRKY75 is S-nitrosylated and stabilised by GSH/GSNO, providing a mechanistic link between redox/NO signalling and transcriptional control of P transport. We propose that elevated GSH under Pi limitation is converted to GSNO, which S-nitrosylates AtWRKY75 and promotes AtPHT1;5 expression, thus driving source-to-sink P reallocation. This pathway integrates redox and NO signalling with transcriptional regulation to maintain P homeostasis under Pi starvation. Summary statementGlutathione-S-nitrosoglutathione module regulates phosphate translocation in Arabidopsis under phosphate starvation via transcriptional activation of AtPHT1;5 gene by AtWRKY75 transcription factor.

plant biology↗

bioRxiv2021

Glutathione regulates subcellular iron homeostasis via transcriptional activation of iron responsive genes in Arabidopsis

While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38 and AtbHLH101 were identified to interact with the promoters of these genes which were in turn activated via S-nitrosylation. Together, the present study delineates the role of GSH-GSNO module in regulating subcellular Fe homeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4 and AtPIC1 via S-nitrosylation of the bHLH TFs during Fe deficiency. Summary statementGlutathione regulates subcellular iron homeostasis under iron deficiency via GSNO dependent transcriptional activation of AtNRAMP3, AtNRAMP4 and AtPIC1 genes by S-nitrosylation of the iron responsive bHLH transcription factors, AtbHLH29, AtbHLH38 and AtbHLH101.

plant biology↗

bioRxiv2020

Spatial distribution of the lectin protein Osr40g3 determines its dual regulatory function in imparting salinity tolerance while impeding seed development in rice

Under changing environmental conditions, salt stress has posed a severe threat to agriculture. Although the R40 family lectins are known to be associated with osmotic stress response, their mechanism of action remains elusive. Among them, Osr40g3 displays the highest expression under salt stress. Here, we report that the constitutive overexpression of Osr40g3 imparts salt tolerance but displays pollen sterility and poor seed development in rice. Promoter analysis and gene expression studies revealed that the gene follows a precise tissue-specific expression pattern, which is essential for proper seed development. Overexpressing the gene under the control of its native promoter rescued the pollen-sterile phenotype while significantly improving salt tolerance. Protein-protein interaction studies demonstrated that Osr40g3 positively regulates an expansin protein, OsEG45, while decreasing the stability of a 14-3-3 protein, OsGF14e. Correspondingly, the OsEG45 overexpression and OsGF14e silencing lines display a salt-tolerant phenotype. Again, silencing OsEG45 in the background of OsGF14e silencing lines resulted in a salt-sensitive phenotype, indicating that salt tolerance of the OsGF14e silencing lines is OsEG45-dependent. Notably, the OsGF14e gene displays early salt responsiveness, while Osr40g3 and OsEG45 display a late response, indicating a spatio-temporal regulation of these genes. Interestingly, constitutive overexpression of Osr40g3 or silencing of OsGF14e leads to diminished gibberellic acid (GA) accumulation that activates the OsEG45 gene. Together, our study demonstrates that during salt stress, Osr40g3, a late salt-responsive gene, confers salt tolerance by negatively regulating OsGF14e while positively regulating OsEG45 via a GA-mediated pathway. This mechanistic insight broadens our understanding of lectin-mediated regulation of salt tolerance.

plant biology↗

bioRxiv2020

Rice lectin protein Osr40c1 imparts drought tolerance by modulating OsSAM2, OsSAP8 and chromatin-associated proteins

Lectin proteins play an important role in biotic and abiotic stress responses in plants. Although the rice lectin protein, Osr40c1, has been reported to be regulated by drought stress, the mechanism of its drought tolerance activity has not been studied so far. In this study, it has been depicted that expression of Osr40c1 gene correlates with the drought tolerance potential of various rice cultivars. Transgenic rice plants overexpressing Osr40c1 were significantly more tolerant to drought stress over the wild-type plants. Furthermore, ectopic expression of the Osr40c1 gene in tobacco yielded a similar result. Interestingly, the protein displayed a nucleo-cytoplasmic localization and was found to interact with a number of drought-responsive proteins like OsSAM2, OsSAP8, OsMNB1B, and OsH4. Fascinatingly, silencing of each of these protein partners led to drought susceptibility in the otherwise tolerant Osr40c1 expressing transgenic tobacco lines indicating that these partners were crucial for the Osr40c1-mediated drought tolerance in planta. Together, the present investigation delineated the novel role of Osr40c1 protein in imparting drought tolerance by regulating the chromatin proteins, OsMNB1B and OsH4, which presumably enables OsSAP8 to induce downstream gene expression. In addition, its interaction with OsSAM2 might induce polyamine biosynthesis thus further improving drought tolerance in plants. HighlightsA rice lectin protein, Osr40c1, plays a crucial role in imparting drought stress tolerance in plants by modulating OsSAM2 as well as the transcriptional regulators OsSAP8, OsMNB1B and OsH4.

plant biology↗

bioRxiv2020

Jacalin domain-containing protein OsSalT interacts with OsDREB2A and OsNAC1 to impart drought stress tolerance in planta

With the changing climatic conditions, drought has become one of the most threatening abiotic stress factors that adversely affect rice cultivation and productivity. Although the involvement of the jacalin domain-containing protein, OsSalT, has been reported in drought and salinity tolerance, its functional mechanism still remains largely unexplored. In this study, expression of the OsSalT gene was found to be positively correlated with the drought tolerance potential with its higher transcript abundance in the tolerant indica rice cultivar, Vandana and lower abundance in the susceptible cultivar, MTU1010. Moreover, the ectopic expression of OsSalT in tobacco imparted drought stress tolerance in the transgenic lines. The transgenic lines exhibited significantly improved growth and higher osmolyte accumulation over the wild-type (WT) plants together with the induction in the OsSalT expression under drought stress. Fascinatingly, the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses confirmed the interaction of OsSalT protein with two interesting transcription factors (TFs), OsNAC1 and OsDREB2A. In silico analysis further revealed that the OsSalT protein interacted with the regulatory domain of OsDREB2A and the C-terminal domain of OsNAC1 leading to their activation and induction of their downstream drought-responsive genes. Together, this study unravels a novel model for OsSalT-mediated regulation of drought tolerance in plants.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗